1. Field of the Invention
The present invention relates to a purified enzymatic preparation derived from Chromobacterium violaceum, having a dehydrogenase activity, to a method for preparing it and to its use in the production of .alpha.,.beta.-dehydrotryptophanyl peptides.
2. Discussion of the Background
A dehydrogenase activity, which acts on the side chain of tryptophan, has been described in Methods in Enzymology (1987, 142, 195-217; K. TAKAI and O. HAYAISHI). Tryptophan side chain oxidase (TSO), which is isolated from Pseudomonas XA (ATCC 29574), is a multi-enzymatic system which catalyses a dehydrogenation of tryptophanyl residues in the C.sub..alpha. and C.sub..beta. positions. However, the unsaturated compound (--.DELTA.Trp--) is only a by-product of the catabolism of tryptophan which is formed in equilibrium with the degradation products (.beta.-hydroxy and .beta.-keto) from a single indolyloxazoline intermediate and does not enable the production of dehydro compounds to be controlled satisfactorily because of the variation in the yield of synthesis of these compounds, especially as a function of the nature of the substrate and the experimental conditions (the pH in particular). Furthermore, this enzyme has a low specificity and converts L-tryptophan (L-Trp), D-tryptophan (D-Trp), residues whose indole ring is modified or substituted (5 OH-Trp) and decarboxylated residues (tryptamine).
P. J. DAVIS et al. have demonstrated the existence of a biotransformation of N-benzyloxycarbonyl-L-tryptophan in a culture of the Gram-negative bacterium, Chromobacterium violaceum (ATCC 12472), which also results in the formation of a double bond between the C.sub..alpha. and C.sub..beta. carbons of L-tryptophan (BBA, 1975, 385, 133-144); they also studied the conversion of indole-3-propionic acid to indole-3-acrylic acid (J. Bacteriol., 1976, 126, 1, 544-546) and the stereochemistry of the dehydrogenation of the side chain of N-benzyloxycarbonyl-(S)-tryptophan (J.C.S. Chem. Comm., 1977, 821, 842-843).
However, this reaction has been demonstrated only on specific substrates whose only common characteristic is the presence of an indole ring, and this only during incubation of resting cells and in bacteria cultures.
It follows from the above that both the reactions described in TAKAI et al. and those described in DAVIS et al. do not permit their use in the production of pure dehydropeptides and dehydroproteins.